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Synergy between sublethal doses of shikonin and metformin fully inhibits breast cancer cell migration and reverses epithelial-mesenchymal transition

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Abstract

Background

Shikonin is a natural multipotent anti-tumorigenic compound. We investigated potential synergy between shikonin and anti-diabetic metformin against tumorigenic properties of breast cancer cell line MCF-7.

Methods and results

The IC50 of shikonin and metformin was determined after a single treatment of two cell lines MCF-7 and MDA-MB-231. We then measured optimal doses of each drug, used in combination, in MCF-7 cells. These sub-IC50 doses were co-applied for all subsequent combined treatments to evaluate their synergistic effects on MCF-7 tumorigenic properties. Next, we examined expression levels of the genes crucial for apoptosis, cell growth, and EMT using RT-PCR or real-time PCR and monitored CD44/CD24 ratios using flow cytometry. Binding energies between shikonin and growth molecules were measured by in silico simulation.

Shikonin caused significantly reduced cell survival that was accelerated by the synergizing presence of metformin. Drug combination induced apoptosis and ROS levels while fully blocking cell migration and reverting EMT. RT-PCR showed strong suppression of BCL-2 but induction of BAX and PTEN. Prolonged shikonin treatment caused a total loss of the nuclear membrane, whereas metformin prevented this damage while promoting apoptotic morphologies. Our real-time PCR detected reduced levels of EMT genes but increases in the anti-EMT gene CDH1. Combined treatment also reduced CD44/CD24 ratios in favor of chemosensitivity. Binding energies strongly favored shikonin interactions with growth-signaling molecules.

Conclusions

Shikonin and metformin synergize in inhibiting the tumorigenic activities of MCF-7 cells including their proliferation, invasiveness, and EMT with a potential to inhibit multidrug resistance.

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Data availability

Any materials and data are available from the corresponding author on reasonable request.

Code availability

Not applicable.

References

  1. Breast Cancer Treatment. National Cancer Institute. Updated 8 Apr, 2021. https://www.cancer.gov/types/breast/patient/breast-treatment-pdq#_125. Accessed 5 Jan 2022

  2. DeSantis CE, Fedewa SA, Goding Sauer A, Kramer JL, Smith RA, Jemal A (2016) Breast cancer statistics, 2015: convergence of incidence rates between black and white women. CA Cancer J Clin 66:31–42

    Article  Google Scholar 

  3. U.S. Breast Cancer Statistics. https://www.breastcancer.org/symptoms/understand_bc/statistics. Accessed 5 Jan 2022

  4. Burrell RA, Swanton C (2014) Tumour heterogeneity and the evolution of polyclonal drug resistance. Mol Oncol 8:1095–1111. https://doi.org/10.1016/j.molonc.2014.06.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Vasan N, Baselga J, Hyman DM (2019) A view on drug resistance in cancer. Nature 575:299–309. https://doi.org/10.1038/s41586-019-1730-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ichihashi N, Kitajima Y (2001) Chemotherapy induces or increases expression of multidrug resistance-associated protein in malignant melanoma cells. Br J Dermatol 144:745–750. https://doi.org/10.1046/j.1365-2133.2001.04129.x

    Article  CAS  PubMed  Google Scholar 

  7. Shibue T, Weinberg RA (2017) EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol 14:611–629. https://doi.org/10.1038/nrclinonc.2017.44

    Article  PubMed  PubMed Central  Google Scholar 

  8. Liu Y, Li Q, Zhou L et al (2016) Cancer drug resistance redox resetting renders a way. Oncotarget 7:42740–42761

    Article  Google Scholar 

  9. Phi LTH, Sari IN, Yang Y-G et al (2018) Cancer Stem Cells (CSCs) in drug resistance and their therapeutic implications in cancer treatment. Stem Cells Int 2018:5416923. https://doi.org/10.1155/2018/5416923

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Li S, LI Q, (2014) Cancer stem cells and tumor metastasis. Int J Oncol 44:1806–1812. https://doi.org/10.3892/ijo.2014.2362

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhao Y, Bao Q, Renner A et al (2011) Cancer stem cells and angiogenesis. Int J Dev Biol 55:477–482. https://doi.org/10.1387/ijdb.103225yz

    Article  CAS  PubMed  Google Scholar 

  12. Basmadjian C, Zhao Q, Bentouhami E, Djehal A, Nebigil CG, Johnson RA, Serova M, de Gramont A, Faivre S, Raymond E, Désaubry LG (2014) Cancer wars: natural products strike back. Front Chem 2:20. https://doi.org/10.3389/fchem.2014.00020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhan T, Rindtorff N, Betge J et al (2019) CRISPR/Cas9 for cancer research and therapy. Semin Cancer Biol 55:106–119. https://doi.org/10.1016/j.semcancer.2018.04.001

    Article  CAS  PubMed  Google Scholar 

  14. Yuan R, Hou Y, Sun W et al (2017) Natural products to prevent drug resistance in cancer chemotherapy: a review. Ann N Y Acad Sci 1401:19–27. https://doi.org/10.1111/nyas.13387

    Article  PubMed  Google Scholar 

  15. Guo C, He J, Song X et al (2019) Pharmacological properties and derivatives of shikonin—A review in recent years. Pharmacol Res 149:104463. https://doi.org/10.1016/j.phrs.2019.104463

    Article  CAS  PubMed  Google Scholar 

  16. Dowling RJ, Goodwin PJ, Stambolic V (2011) Understanding the benefit of metformin use in cancer treatment. BMC Med 9:33. https://doi.org/10.1186/1741-7015-9-33

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kasibhatla S, Amarante-Mendes GP, Finucane D, Brunner T, Bossy-Wetzel E, Green DR (2006) Acridine Orange/ethidium bromide (AO/EB) staining to detect apoptosis. CSH Protoc. https://doi.org/10.1101/pdb.prot4493

    Article  PubMed  Google Scholar 

  18. Eruslanov E, Kusmartsev S (2010) Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol 594:57–72. https://doi.org/10.1007/978-1-60761-411-1_4

    Article  CAS  PubMed  Google Scholar 

  19. Gardaneh M, Gholami M, Maghsoudi N (2011) Synergy between glutathione peroxidase-1 and astrocytic growth factors suppresses free radical generation and protects dopaminergic neurons against 6-hydroxydopamine. Rejuvenation Res 14:195–204. https://doi.org/10.1089/rej.2010.1080

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262

    Article  CAS  PubMed  Google Scholar 

  21. Gardaneh M, Nayeri Z, Akbari P et al (2020) Molecular simulations identify target receptor kinases bound by astaxanthin to induce breast cancer cell apoptosis. Arch Breast Cancer. https://doi.org/10.32768/abc.20207272-82

    Article  Google Scholar 

  22. Wang T, Narayanaswamy R, Ren H, Torchilin VP (2016) Combination therapy targeting both cancer stem-like cells and bulk tumor cells for improved efficacy of breast cancer treatment. Cancer Biol Ther 17:698–707. https://doi.org/10.1080/15384047.2016.1190488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Fiorillo M, Tóth F, Sotgia F, Lisanti MP (2019) Doxycycline, Azithromycin and Vitamin C (DAV): A potent combination therapy for targeting mitochondria and eradicating cancer stem cells (CSCs). Aging (Albany NY) 11:2202–2216

    Article  CAS  Google Scholar 

  24. Li W, Liu J, Jackson K et al (2014) Sensitizing the therapeutic efficacy of taxol with shikonin in human breast cancer cells. PLoS ONE 9:e94079. https://doi.org/10.1371/journal.pone.0094079

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Goldman RD, Shumaker DK, Erdos MR et al (2004) Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci 101:8963–8968. https://doi.org/10.1073/pnas.0402943101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lopez-Mejia IC, Vautrot V, De Toledo M et al (2011) A conserved splicing mechanism of the LMNA gene controls premature aging. Hum Mol Genet 20:4540–4555. https://doi.org/10.1093/hmg/ddr385

    Article  CAS  PubMed  Google Scholar 

  27. Egesipe AL, Blondel S, Lo Cicero A, Jaskowiak AL, Navarro C, Sandre-Giovannoli A, Levy N, Peschanski M, Nissan X (2016) Metformin decreases progerin expression and alleviates pathological defects of Hutchinson-Gilford progeria syndrome cells. NPJ Aging Mech Dis 2:16026. https://doi.org/10.1038/npjamd.2016.26

    Article  PubMed  PubMed Central  Google Scholar 

  28. Yeh YC, Liu TJ, Lai HC (2015) Shikonin induces apoptosis, necrosis, and premature senescence of human A549 lung cancer cells through upregulation of p53 expression. Evid Based Complement Alternat Med 2015:620383. https://doi.org/10.1155/2015/620383

    Article  PubMed  PubMed Central  Google Scholar 

  29. Wang F, Mayca Pozo F, Tian D et al (2020) Shikonin inhibits cancer through P21 upregulation and apoptosis induction. Front Pharmacol 11:861. https://doi.org/10.3389/fphar.2020.00861

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Bao C, Liu T, Qian L et al (2021) Shikonin inhibits migration and invasion of triple-negative breast cancer cells by suppressing epithelial-mesenchymal transition via miR-17-5p/PTEN/Akt pathway. J Cancer 12:76–88. https://doi.org/10.7150/jca.47553

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wiench B, Eichhorn T, Paulsen M, Efferth T (2012) Shikonin directly targets mitochondria and causes mitochondrial dysfunction in cancer cells. Evidence-Based Complement Altern Med 2012:726025. https://doi.org/10.1155/2012/726025

    Article  Google Scholar 

  32. Verma NK, Dourlat J, Davies AM et al (2009) STAT3-Stathmin interactions control microtubule dynamics in migrating t-cells. J Biol Chem 284:12349–12362. https://doi.org/10.1074/jbc.M807761200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Trinh SX, Nguyen HTB, Saimuang K et al (2017) Metformin inhibits migration and invasion of cholangiocarcinoma cells. Asian Pac J Cancer Prev 18:473–477

    PubMed  Google Scholar 

  34. Cao HH, Liu DY, Lai YC, Chen YY, Yu LZ, Shao M, Liu JS (2020) Inhibition of the STAT3 signaling pathway contributes to the anti-melanoma activities of shikonin. Front Pharmacol 11:748. https://doi.org/10.3389/fphar.2020.00748

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Schexnayder C, Broussard K, Onuaguluchi D, Poché A, Ismail M, McAtee L, Llopis S, Keizerweerd A, McFerrin H, Williams C (2018) Metformin inhibits migration and invasion by suppressing ROS production and COX2 expression in MDA-MB-231 breast cancer cells. Int J Mol Sci 19(11):3692. https://doi.org/10.3390/ijms19113692

    Article  CAS  PubMed Central  Google Scholar 

  36. Chang I-C, Huang Y-J, Chiang T-I et al (2010) Shikonin induces apoptosis through reactive oxygen species/extracellular signal-regulated kinase pathway in osteosarcoma cells. Biol Pharm Bull 33:816–824. https://doi.org/10.1248/bpb.33.816

    Article  CAS  PubMed  Google Scholar 

  37. Zhang Z, Zhang Z, Li Q et al (2017) Shikonin induces necroptosis by reactive oxygen species activation in nasopharyngeal carcinoma cell line CNE-2Z. J Bioenerg Biomembr 49:265–272. https://doi.org/10.1007/s10863-017-9714-z

    Article  CAS  PubMed  Google Scholar 

  38. Algire C, Moiseeva O, Deschênes-Simard X et al (2012) Metformin reduces endogenous reactive oxygen species and associated DNA damage. Cancer Prev Res 5:536–543. https://doi.org/10.1158/1940-6207.CAPR-11-0536

    Article  CAS  Google Scholar 

  39. Marinello PC, Panis C, Silva TNX et al (2019) Metformin prevention of doxorubicin resistance in MCF-7 and MDA-MB-231 involves oxidative stress generation and modulation of cell adaptation genes. Sci Rep 9:5864. https://doi.org/10.1038/s41598-019-42357-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Li W, Ma H, Zhang J, Zhu L, Wang C, Yang Y (2017) Unraveling the roles of CD44/CD24 and ALDH1 as cancer stem cell markers in tumorigenesis and metastasis. Sci Rep 7(1):13856. https://doi.org/10.1038/s41598-017-14364-2.Erratum.In:SciRep.2018Mar6;8(1):4276

    Article  PubMed  PubMed Central  Google Scholar 

  41. Toole BP (2009) Hyaluronan-CD44 interactions in cancer: paradoxes and possibilities. Clin Cancer Res 15:7462–7468. https://doi.org/10.1158/1078-0432.CCR-09-0479

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Misra S, Ghatak S, Toole BP (2005) Regulation of MDR1 expression and drug resistance by a positive feedback loop involving hyaluronan, phosphoinositide 3-kinase, and ErbB2. J Biol Chem 280:20310–20315. https://doi.org/10.1074/jbc.M500737200

    Article  CAS  PubMed  Google Scholar 

  43. Gilg AG, Tye SL, Tolliver LB et al (2008) Targeting Hyaluronan Interactions in Malignant Gliomas and Their Drug-Resistant Multipotent Progenitors. Clin Cancer Res 14:1804–1813. https://doi.org/10.1158/1078-0432.CCR-07-1228

    Article  CAS  PubMed  Google Scholar 

  44. Chen C, Zhao S, Karnad A, Freeman JW (2018) The biology and role of CD44 in cancer progression: therapeutic implications. J Hematol Oncol 11:64. https://doi.org/10.1186/s13045-018-0605-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Han W, Li L, Qiu S et al (2007) Shikonin circumvents cancer drug resistance by induction of a necroptotic death. Mol Cancer Ther 6:1641–1649. https://doi.org/10.1158/1535-7163.MCT-06-0511

    Article  CAS  PubMed  Google Scholar 

  46. Wu H, Xie J, Pan Q et al (2013) Anticancer agent shikonin is an incompetent inducer of cancer drug resistance. PLoS ONE 8:e52706. https://doi.org/10.1371/journal.pone.0052706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Coyle C, Cafferty FH, Vale C, Langley RE (2016) Metformin as an adjuvant treatment for cancer: a systematic review and meta-analysis. Ann Oncol 27:2184–2195. https://doi.org/10.1093/annonc/mdw410

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This study was financially supported by a grant from National Institute of Genetic Engineering and Biotechnology (NIGEB) (Grant 589).

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Authors and Affiliations

  1. National Institute of Genetic Engineering and Biotechnology, HWY Kilometer 15, PO BOX 14965/161, Karaj, Tehran, Iran

    Abolfazl Rostamian Tabari, Pegah Gavidel, Farzaneh Sabouni & Mossa Gardaneh

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  1. Abolfazl Rostamian Tabari
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  2. Pegah Gavidel
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  3. Farzaneh Sabouni
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  4. Mossa Gardaneh
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Contributions

ART carried out most of the experiments. PG designed and implemented a part of experiments including invasion and cell co-staining plus molecular docking. FS contributed to the dose determination of shikonin and metformin as well as editing and reviewing the manuscript. MG was a grantee of the project, supervised the first author, and designed the whole structure of the manuscript in detail.

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Correspondence to Mossa Gardaneh.

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Tabari, A.R., Gavidel, P., Sabouni, F. et al. Synergy between sublethal doses of shikonin and metformin fully inhibits breast cancer cell migration and reverses epithelial-mesenchymal transition. Mol Biol Rep 49, 4307–4319 (2022). https://doi.org/10.1007/s11033-022-07265-9

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